Misregulation of the ubiquitin-proteasome system (UPS) and Endoplasmic Reticulum-Associated Degradation (ERAD) is characteristic of many human diseases, including cancer, neurodegenerative disorders, cystic fibrosis, and diabetes (1). Therapeutic interventions targeting the UPS remain underdeveloped. The yeast transmembrane ubiquitin ligase Doa10, conserved in humans, catalyzes ubiquitination of ERAD substrates and regulatory nuclear proteins (2). Doa10 accesses its substrates by localizing to both the inner nuclear membrane (INM) and ER (3). The broad objectives of the proposed research are to investigate factors governing the localization of Doa10 and the manner by which Doa10 and Hex3-Slx8, a ubiquitin ligase with overlapping specificity, interact with their nuclear substrates. The specific aims of this proposal are to characterize the determinants and consequences of Doa10 localization to the INM and investigate the action of Doa10 and Hex3-Slx8 on DNA-bound substrates. Biochemical and cell- biological approaches will be employed to accomplish these aims. A nuclear localization signal (NLS) (required for the localization of another INM protein (4)) will be appended to wild-type Doa10 and a bulky Doa10 fusion protein that cannot normally gain access to the nucleus, presumably due to a size restriction on unassisted nuclear entry (3). Localization of the fusion proteins will be monitored, and the consequences of altered localization for Doa10 activity toward its nuclear and ER-associated substrates will be assessed. It will be determined if the NLS-modified proteins require an active nuclear transport system for nuclear entry, as do soluble NLS-bearing proteins. Second, novel ChlP-seq methodology will be used to determine the chromosomal loci with which Doa10 and Hex3-Slx8 interact. The requirements and consequences for these interactions will be characterized. This strategy may also enable discovery of novel Doa10 and Hex3-Slx8 substrates. These experiments are highly likely to provide a foundation for therapeutic advances for UPS- associated conditions, consistent with the mission of the National Institute of General Medical Sciences. Public Health Relevance: Many human diseases, such as specific forms of cancer, neurodegenerative disorders, cystic fibrosis, and diabetes, are associated with malfunctioning of the cellular system required for the destruction of proteins in human cells (1). The work proposed here will improve the understanding of an important enzyme in yeast called Doa10 (which is closely related to a human enzyme of similar function) that is critically involved in protein destruction. These studies are likely to assist in the development of new medical treatments for patients with diseases of aberrant protein destruction.